Cerenkov radiation is a phenomenon in which optical photons are emitted when a charged particle moves faster than the speed of light in a dielectric medium such as tissue. Recently we, and others, have discovered that measurable visible light due to the Cerenkov effect is produced in vivo following administration of ?-emitting radionuclides to small animals. Furthermore, the amounts of injected activity required to produce a detectable signal are consistent with small animal molecular imaging applications. This observation has led to the development of a new hybrid molecular imaging modality known as Cerenkov luminescence imaging (CLI) that allows the spatial distribution of biomolecules labeled with ?-emitting radionuclides to be imaged in vivo using sensitive charge-coupled device (CCD) cameras. This is especially valuable for preclinical studies with important therapeutic ?-emitting radionuclides, such as 90Y, that cannot readily be imaged at tracer doses using any other in vivo imaging technique. CLI also provides a fast, low-cost, readily accessible approach to image PET radiotracers in small animals. Translational opportunities for Cerenkov luminescence also exist. CLI might be possible for skin, eye and oral cavity, or using endoscopic or catheter-based devices inside the body. Another possible application that is being explored is to use the abundant blue/UV Cerenkov light produced by radiotracers as a source of internal light delivery deep inside tissues for photoactivation, for example in photodynamic therapy and photoactivated drug delivery. The goal of this proposal is to provide a quantitative understanding of Cerenkov luminescence as it applies to biomedical applications and to produce data that serves as a platform for guiding the development of small-animal and translational Cerenkov applications by the molecular imaging and therapeutics community. The specific aims address obtaining a detailed quantitative understanding of Cerenkov signals and spectra that can be measured from tissues through a combination of computation/simulation, phantom experiments and in vivo studies. We will compare the performance of two different camera technologies for the detection of weak Cerenkov signals, and determine detection limits. The ability to exploit spectral information for Cerenkov tomography, and for spectral unmixing in multimodal studies that involve both Cerenkov and bioluminescence signals, also will be characterized. Finally, we will estimate the light energy delivered to tissues by radiotracers via the Cerenkov effect, to determine if this mechanism can feasibly be used as a source of internal excitation for photoactivation. The anticipated outcome of this work is a detailed understanding of the production, distribution and transport of visible Cerenkov radiation by systemically-administered radiotracers, that will serve as a critical foundation on which to develop biomedical applications based on this newly-observed phenomenon.